Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. My current research uses the genetically-tractable bacterium Bacillus subtilis as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. I have been characterizing the protein ConE (formerly YddE) which is encoded on the B. subtilis conjugal element ICEBs1. ConE is related to proteins encoded on conjugal elements in numerous bacteria, including the Gram-positive pathogens S. aureus, C. difficile, and L. monocytogenes. ConE belongs to a large superfamily of ATP-dependent pumps involved in the extrusion of proteins and DNA through membrane pores. I have shown that ConE and its ATPase domain are essential for mating of ICEBs1. In addition, ConE-GFP localizes at the cell poles, in close association with the membrane (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, I propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. I plan on analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques.

Current Members of the Berkmen Lab

Stephanie and Bridget at their poster at the National ACS Meeting in San Francisco, March 2010

Bridget Giarusso - Biochemistry Major
As a research assistant, Bridget and Stephanie used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. Since His6-ConE is functional for mating, we can purify His6-ConE and begin in vitro assays to determine whether it can bind ATP. She presented this research with Stephanie at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She is also helping to clone several ICEBs1 genes.

Stephanie Laurer - Biochemistry Major
As a research assistant, Stephanie and Bridget used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. Since His6-ConE is functional for mating, we can purify His6-ConE and begin in vitro assays to determine whether it can bind ATP. She presented this research with Bridget at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She is also helping to clone several ICEBs1 genes. On the side, Stephanie has had an interest in genetically modified food. During her first year at Suffolk, she used a PCR-based assay to detect the Bt gene in corn. She found that at least half of the samples she tested (6 of 11) were genetically modified. She presented this work at the 2009 Suffolk science banquet where her poster won first place.

Former Members of the Berkmen Lab

Matt Hamada - B.S. Biochemistry, December 2010
For CHEM L333, Matt cloned the yddC gene encoded on the ICEBs1 conjugal element. For CHEM L428/L429, Matt will be using fluorescence microscopy to determine whether yddC and other ICEBs1 genes are required for ConE to localize at the cell poles. He presented his research with Cori at the 2009 Boston Bacterial Meeting.

Cori Leonetti - B.S. Biochemistry, May 2010
For CHEM L333, Cori helped clone the yddB gene encoded on the ICEBs1 conjugal element. Cori is extending her CHEM L333 project for CHEM L428/L429. She will be testing whether yddB and other ICEBs1 genes are required for mating. Cori presented her research with Matt at the 2009 Boston Bacterial Meeting.

Erin Cross - B.S. Biochemistry, May 2009
In CHEM L333, Erin and her class mates attempted to clone various C-terminal truncations of YddE fused to GFP. For CHEM L428/L429, she used fluorescence microscopy to analyze what parts of YddE are required for localization to the cell poles. She found that the C-terminal half of YddE is critical for localization. In addition, she found the YddE-GFP localizes at the cell poles, even when the upstream gene yddD is not expressed in cis. She presented this work at the national ACS meeting in March 2009 and at the 2009 Suffolk Science Banquet where her poster won 3rd place.

Maria Levicheva - B.S. Biochemistry, Honors Program, May 2009
For CHEM L333, Maria began construction of a his-tagged YddE. For CHEM L428/L429, she purified and characterized His6-YddE to enable future students to perform ATPase assays to determine whether YddE can hydrolyze ATP in vitro. She presented this work at the national ACS meeting in March 2009.

Tamara Wong - B.S. Biochemistry Forensic Science, May 2009
Tamara cloned the His6-YddE construct so that we can test whether this protein can support mating. In addition, Tamara purified His6-YddE to enable future ATPase assays.

Emma-Kate Loveday - B.S. Biochemistry, May 2008
For her CHEM L428/L429 project, Emma-Kate constructed two variants of YddE and tested their effects on mating. She found that the Walker B (ATP hydrolysis domain) of YddE is essential for mating. She also found that the N-terminus of YddE does not contribute significantly to mating. She presented her work at the Boston Bacterial Meeting in June 2008 and the Cold Spring Harbor Molecular Genetics of Bacteria and Phages Meeting in August 2008. Emma is now a graduate student in the microbiology and immunology Ph.D. program at the University of British Columbia supported by a prestigious NSF Fellowship.

Berkmen MB and Grossman AD. (2007) Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol, 63(1): 150-165.